OVERVIEW
Phospholipase C
Phospholipase C (PLC) belongs to a family of enzymes, also known as disulfide isomerases, which play a very important role in transmembrane signal transduction. Many extracellular signaling molecules including hormones, growth factors, neurotransmitters, and immunoglobulins bind to their respective cell surface receptors and activate PLCs. The role of an activated PLC is to catalyze the hydrolysis of phosphatidyl-inositol-4, 5-bisphosphate (PIP2), a minor component of the plasma membrane to produce diacylglycerol and inositol 1, 4, 5-trisphosphate (IP3).
In their respective biochemical pathways, IP3 and diacylglycerol serve as second messengers and trigger a series of intracellular responses. IP3 induces the release of Ca.sup.++ from internal cellular storage, and diacylglycerol activates protein kinase C (PKC). Both pathways are part of transmembrane signal transduction mechanisms which regulate cellular processes which include secretion, neural activity, metabolism, and proliferation. For example, interleukin 4 receptor signaling in human monocytes involves activation of PLC (Ho, J L et al. (1994) J Exper Med 180: 1457-69).
Several distinct isoforms of PLC have been identified and are categorized as PLC-beta, PLC-gamma, and PLC-delta. Subtypes are designated by adding Arabic numbers after the Greek letters, e.g. PLC-beta-1. PLCs have a molecular mass of 62-68 kDa, and their amino acid sequences show two regions of significant similarity. The first region designated X has about 170 amino acids, and the second or Y region contains about 260 amino acids.
The Mechanism of G Protein-mediated Transmembrane Signaling
The activation of a particular PLC is mediated by a guanine nucleotide binding-regulatory protein (G-protein) or by the intrinsic tyrosine kinase activity of cell surface receptors. Many plasma membrane-bound receptors, including the hormone receptors, activate the cell's G proteins. Each G protein can act as a molecular switch turning on one or more membrane-bound effectors such as adenylate cyclase, ion channels, or phospholipase C.
G proteins are heterotrimers with alpha, beta, and gamma subunits. Inactive G proteins have guanine diphosphate (GDP) molecule bound tightly to their alpha subunit. When a G-protein linked receptor binds an extracellular ligand, such as a hormone, the hormone-receptor complex causes dissociation of GDP from the alpha-subunit. Immediately thereafter, GTP molecules fill the site, and activity of the alpha subunit's intrinsic ATPase causes dissociation of the G protein from the hormone-receptor complex. Simultaneously, GTP binding reduces the affinities between the alpha-, beta- and gamma-subunits and frees the beta-gamma complex. In some systems, the beta-gamma complex then activates PLC beta-2 (Katz A et al (1992) Nature 360:686-9).
Phospholipase Isoforms and Their Cellular Activity
The catalytic activities of the three isoforms of PLC are dependent upon Ca.sup.++. It has been suggested that the binding sites for Ca.sup.++ in the PLCs are located in the Y-region, one of two conserved regions. The hydrolysis of common inositol-containing phospholipids--phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4, 5-bisphosphate (PIP2) by any of the isoforms yields cyclic and noncyclic inositol phosphates. A large number of hormones and related molecules are known to activate phospholipases.
PLC-beta Isoforms
Both beta-1 and beta-2 isoforms of PLC are activated by certain subtypes of G-proteins and related G protein alpha-subunits during the transduction of signals from cell surface receptors to PLC. There may be two distinct types of G proteins, one pertussis toxin sensitive and the other insensitive, which activate the beta-1 isoform. Katz A et al (supra) suggest that the beta subunit of the G protein may also activate PLC beta-1. The activation of PLCs is achieved by increasing their intrinsic activity rather than by reducing the PLC's requirement of Ca.sup.++ in the cytosol.
PLC-gamma Isoforms
The PLC gamma-1 isoform is mainly phosphorylated and activated by growth factor receptors belonging to the tyrosine kinase family. In addition, the growth factor receptors associate with the gamma-1 isoform before any tyrosine phosphorylation occurs. The major sites of tyrosine phosphorylation by the receptors for epidermal growth factor (EGF), platelet-derived growth factor. (PDGF), and nerve growth factor (NGF) appear to be Tyr-771, Tyr-783, and Tyr-1254 in the PLC amino acid sequence. Phosphorylation of Tyr-783 in the gamma-1 isoform by the receptor tyrosine kinase is essential for the activation of the gamma-1 isoform.
Other evidence suggests that non-receptor protein tyrosine kinases can also phosphorylate and activate the gamma-1 isoform in response to certain cell surface receptors in leukocytes. For instance, the T cell antigen receptors complex can recognize antigens and transduce signals across the plasma membrane. Likewise, it appears that non-receptor protein tyrosine kinases can activate the gamma-2 isoform.
Evidence suggests that the activation of the gamma-2 isoform is necessary for stimulation of phospholipase D by platelet-derived growth factor (Yeo E-J et al (1994) J Biol Chem 269(45):27823-27826). Other evidence suggests that B cell surface antigen CD20 is associated with tyrosine and serine kinases and involved in tyrosine phosphorylation and activation of the gamma-1 and gamma-2 isoforms (Deans J P et al (1993) J Immunol 151 (9):4494-4504).
Growth factor-induced activation of PLCs appears to be independent of G-protein mediation. Marrero M B et al (1994, J Biol Chem 269:10935-39), however, reported an exception in rat aortic vascular smooth muscle cells where PLC-gamma-1 was activated by a G-protein-coupled receptor.
PLC-delta
Neither receptors nor transducers of the PLC-delta isoforms have been identified.
Inhibition of PLCs by Protein Kinases
Evidence suggests that the activation of protein kinases may serve as negative feed back signals and attenuate receptor-coupled PLC activity including the magnitude and duration in certain types of cells. For instance, the phosphorylation of Thr-654 in the EGF receptors by protein kinase prevents activation of the gamma-1 isoform by reducing the capacity of the receptor tyrosine kinase to phosphorylate the gamma-1 isoform. In addition, PKC activators such as cAMP and phorbol 12-myristate 13-acetate (PMA) attenuate the PIP2 hydrolysis induced by T cell antigen receptors. Likewise, the beta-1 isoform of PLC appears to be regulated by PKC in certain cells.
Effects of The Second Messengers And Calcium Cations:
Inositol trisphosphate
Once activated, PLCs catalyze the hydrolysis of phosphatidylinositol-4, 5-bisphosphate (PIP2) to produce diacylglycerol and inositol 1, 4, 5-trisphosphate (IP3), all of which serve as second messengers. Inositol trisphosphate releases Ca.sup.++ from intracellular stores and increases the influx of Ca.sup.++ from the extracellular fluid. Ca.sup.++ directly regulates target enzymes and indirectly affects other enzymes by functioning as a second messenger and interacting with Ca.sup.++ -binding proteins, such as troponin C and calmodulin.
Deactivation of the inositol trisphosphate pathway is achieved by active transport of Ca.sup.++ into cells and extrusion of ions by plasma membrane-bound, Ca.sup.++ -pumping ATPases. Likewise, sequential phosphorylation degrades inositol trisphosphate.
Diacylglycerol
Diacylglycerol, a product of the hydrolysis by PLCs, acts as a second messenger by activating protein kinase C. After protein kinase C binds to diacylglycerol, the requirement of Ca.sup.++ by protein kinase C decreases to the level of free Ca.sup.++ found in the cytosol. Activated protein kinase C phosphorylates a great number of intracellular proteins. The termination of the diacylglycerol effect is achieved by enzymatic recycling to form phosphatidylinositol. Alternatively, a diacylglycerol lipase breaks down diacylglycerol.
PLC and Diseases
Evidence indicates that a high percentage of primary human mammary carcinomas concomitantly show increased levels of receptor EGF and PLC-gamma-1. Likewise, studies on spontaneous hypertensive rats have suggested that one of the main causes for the hypertension is an abnormal activation of PLC-delta-1 resulting from point mutations in the X and Y regions of the PLC amino acid sequence.
The biology of PLC is reviewed, inter alia, in Isselbacher K J et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York City; Kuruvilla A et al (1993) J Immun 151:637-648; and Rhee S G and Choi K D (1994) J Biol Chem 267:12393-12396.